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Toxoplasma gondii is a major foodborne pathogen capable of infecting all warm-blooded animals, including humans. Although oocyst-associated toxoplasmosis outbreaks have been documented, the relevance of the environmental transmission route remains poorly investigated. Thus, we carried out an extensive systematic review on T. gondii oocyst contamination of soil, water, fresh produce, and mollusk bivalves, following the PRISMA guidelines. Studies published up to the end of 2020 were searched for in public databases and screened. The reference sections of the selected articles were examined to identify additional studies. A total of 102 out of 3201 articles were selected: 34 articles focused on soil, 40 focused on water, 23 focused on fresh produce (vegetables/fruits), and 21 focused on bivalve mollusks. Toxoplasma gondii oocysts were found in all matrices worldwide, with detection rates ranging from 0.09% (1/1109) to 100% (8/8) using bioassay or PCR-based detection methods. There was a high heterogeneity (I2 = 98.9%), which was influenced by both the sampling strategy (e.g., sampling site and sample type, sample composition, sample origin, season, number of samples, cat presence) and methodology (recovery and detection methods). Harmonized approaches are needed for the detection of T. gondii in different environmental matrices in order to obtain robust and comparable results.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

Toxoplasma gondii is a zoonotic parasite infecting all warm-blooded animals, including humans. The contribution of environmental contamination by T. gondii oocysts to infections is understudied. The aim of the current work was to explore T. gondii serology as a means of attributing the source of infection using a robust stepwise approach. We identified in silico thirty-two promising oocyst-specific antigens from T. gondii ´omics data, recombinantly expressed and purified them and validated whether serology based on these proteins could discriminate oocyst- from tissue cyst-driven experimental infections. For this, three well-characterized serum panels, sampled from 0 to 6 weeks post-infection, from pigs and sheep experimentally infected with T. gondii oocysts or tissue cysts, were used. Candidate proteins were initially screened by Western blot with sera from pigs or sheep, infected for different times, either with oocysts or tissue cysts, as well as non-infected animals. Only the recombinant proteins TgCCp5A and TgSR1 provoked seroconversion upon infection and appeared to discriminate between oocyst- and tissue cyst-driven infections with pig sera. They were subsequently used to develop an enzyme-linked immunosorbent assay test for pigs. Based on this assay and Western blot analyses, a lack of stage specificity and low antigenicity was observed with all pig sera. The same was true for proteins TgERP, TgSporoSAG, TgOWP1 and TgOWP8, previously described as source-attributing antigens, when analyzed using the whole panels of sera. We conclude that there is currently no antigen that allows the discrimination of T. gondii infections acquired from either oocysts or tissue cysts by serological tests. This work provides robust new knowledge that can inform further research and development toward source-attributing T. gondii serology.